The glycoprotein hormones (GpHs), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH), regulate fertility and metabolism. Their receptors (GpHRs) are distinguished from most G protein-coupled receptors by the presence of large N-terminal extracellular hormone-binding domains (ECDs). Each ECD consists of nine leucine-rich repeats (LRRs), flanked by N-terminal and C-terminal cysteine-rich regions (NCR and CCR, respectively). The signal of specific hormone binding to the ECD is conveyed by its CCR to the heptahelical transmembrane region (TM). Mutations, activating or inactivating GpHR function, have been identified in hyperthyroidism, precocious puberty and pseudohermaphroditism; moreover, polymorphic variants of LH and FSH receptors have been shown to change the susceptibility to female infertility, breast cancer and risk of bone fractures. AIMS: 1. Identify common hormone-contact sites and key hormone-selective determinants in LRRs of GpHRs, 2. Identify residues in the cysteine-rich regions of GpHRs important for GpH binding and for conveying the binding signal to the TM, and 3. Determine the three-dimensional structure of GpH-GpHR complexes. METHODS: Domain exchange/site-directed mutagenesis, expression, hormone binding and cAMP-dependent reporter activity as readouts, molecular modeling, ECD protein expression, protein purification, crystallization, crystal structure determination. OUTCOME: The results of the proposed studies will increase our knowledge of the molecular mechanisms by which GpHRs recognize the 'correct' GpH, as well as the step-wise propagation of the signal to the TM following ligand binding. These data, in combination with the mutant 'gain-of-function' GpHRs, will be molded into a general model of GpHR activation, essential for the development of better treatment protocols and the rational design of drugs that change receptor activity. Moreover, these data may yield a better understanding of the etiology of different pathological states known for some GpH-GpHR pairs.